Use of a green fluorescent protein marker for studying splash dispersal of sporangia of Phytophthora infestans

The purpose of this study was to develop a new technique to evaluate the number of spores incorporated in splash droplets by the use of an engineered fluorescent pathogen strain and image analysis hardware and software. The inoculum source consisted of a tomato leaflet infected with a Phytophthora infestans strain transformed with the gene encoding the green fluorescent protein (GFP). Splash droplets formed after impact of incident drops on sporulating lesions were collected on microscope slides located at different distances from the source. Each slide was examined using a fluorescent microscope to visualize the GFP expressing sporangia. Photographs were taken and assessed using image processing to count the sporangia incorporated in each droplet. Data analysis confirmed characteristics of splash dispersal shown using other methods. The use of fluorescent sporangia also facilitates the selective detection and counting of viable (living) sporangia, and is a tool that can be use in the study of splash dispersed diseases